Heterocyclic and carbonate derivatives of NDGA and their use as new anti-HIV and anti-cancer agents

ABSTRACT

Reaction of nordihydroguaiaretic acid with various alkyl chlorides, 1-piperidinecarbonyl chloride, methyl chloroformate, or 1,1′-carbonyldiimidazole under alkaline conditions produced the corresponding phenol ethers, carbamates and carbonates, respectively, in 67-83% yields (Scheme 1 and Scheme 2). Among these derivatives, the nitrogen-containing compounds were converted to the corresponding hydrochloride salts. Having good solubility, these NDGA derivatives were found to be stable in aqueous solution. These new compounds exerted potent activities against HIV Tat-regulated transactivation in cos-7 cells. The most active transcription inhibitor compound of this series 5b (P 4 N, Tetrapiperidino NDGA, meso-2,3-dimethyl-1,4-bis(3,4-[2-(piperidino)ethoxypehnyl])butane tetrakishydrochloride salt) has an IC 50  of 0.88 μ M .

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/783,970, filed Apr. 13, 2007, which claims priority to U.S.Provisional application No. 60/792,332, filed Apr. 17, 2006, both ofwhich are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to nordihydroguaiaretic acid derivativesparticularly useful for treating viral infections and tumors.

BACKGROUND

Nordihydroguaiaretic acid (NDGA, 1) is a lignan found in the leaves andtwigs of the shrub Larrea tridentata. Being a lipoxygenases inhibitor,NDGA can induce cystic nephropathy in the rat.¹ In addition, it showsvarious bioactivities, including inhibition of protein kinase C,²induction of apoptosis,³ alterations of membrane,⁴ elevation of cellularCa²⁺ level⁵ and activation of Ca²⁺ channels in smooth muscle cells,⁶breakdown of pre-formed Alzheimer's beta-amyloid fibrils in vitro,⁷anti-oxidation,⁸ etc. This natural product is used commercially as afood additive to preserve fats and butter in some parts of the world.Recently, the derivatives of the plant lignan NDGA have been used toblock viral replication through the inhibition of viraltranscription.⁹⁻¹⁶ These compounds can inhibit production of HIV,⁹⁻¹³herpes simplex virus,^(14,15) and HPV transcripts¹⁶ by deactivation oftheir Sp1-dependent promoters. Moreover,(tetra-O-methyl)nordihydroguaiaretic acid (M₄N, EM1421 2) can functionas an anti-HIV proviral transcription inhibitor and causes growth arrestof a variety of transformed human and mouse cells in culture and inmice.^(17,18,22) Compound M₄N (EM1421) is currently in clinical trialsagainst human cancers.²³

While M₄N (2) is a strikingly effective and non-toxic anticancer agent,M₄N and several other methylated NDGAs, all show poor water solubilitywhich somewhat limit their applicabilities for certain drug actionstudies. To circumvent this problem, a water soluble derivative of NDGA,(tetra-O-dimethylglycyl)nordihydroguaiaretic acid (G₄N, 4) has beendesigned and synthesized.¹¹ G₄N is a very effective mutation-insensitiveinhibitor to HIV-1, HSV-1 and HSV-2.^(10,15) However, it is somewhatunstable and has a relatively short half-life in aqueous solution,reportedly due to the ester bonds connecting the dimethyl glycinemoieties onto the NDGA main skeleton.¹¹

Therefore, there is a need for NDGA derivatives with improved watersolubility and stability having the desired pharmaceutical effects.Accordingly, we have developed new derivatives of NDGA that have theseadvantages and will be useful in therapeutic compositions and treatmentmethods. We describe herein the chemical synthesis of these newcompounds and their efficacies.

SUMMARY

The present invention provides compounds, pharmaceutical compositions,methods and kits for the treatment of diseases and disorders, inparticular, viral infections, and proliferative diseases such as tumors.Described herein are new compounds of formula

wherein

wherein R₁-R₄ are independently selected from the group consisting of:

(a) a straight chain or branched lower alkyl group substituted with aheterologous nitrogen-containing ring group of five to seven members;

(b) in combination with the O attached to the phenyl ring, a carbamategroup other than a methyl carbamate group, optionally containing a fiveor six membered carbon-nitrogen ring;

(c) in combination with the O attached to the phenyl ring, a C₁-C₆straight chain or branched carbonate group; and

(d) wherein R₁ and R₂; or R₃ and R₄; or R₁ and R₂, and R₃ and R₄;together with the phenyl group to which they are attached, combine toform a cyclic carbonate group other thanmeso-2,3-dimethyl-1,4-bis(benzo[d][1,3]dioxol-2-one)butane;

and salts of the compounds.

Examples of such compounds are

-   meso-2,3-dimethyl-1,4-bis(3,4-[2-(pyrrolidino)ethoxyphenyl])butane,-   meso-2,3-dimethyl-1,4-bis(3,4-[2-(piperidino)ethoxyphenyl])butane,-   meso-2,3-dimethyl-1,4-bis(3,4-[2-(morpholino)ethoxyphenyl])butane,-   meso-2,3-dimethyl-1,4-bis(3,4-[3-(morpholino)propoxyphenyl])butane,-   meso-2,3-dimethyl-1,4-bis[3,4-(phenyl    piperidine-1-carboxylate)]butane,-   meso-2,3-dimethyl-1,4-bis[3,4-(methyl phenyl carbonate)]butane, and-   meso-2,3-dimethyl-1,4-bis(benzo[d][1,3]dioxol-2-one)butane.

Substitutions on the heterologous rings may include halogens, loweralkyl and/or alkoxy groups.

Pharmaceutical compositions described herein contain a substantiallypure preparation of at least one such NDGA derivative in apharmaceutically acceptable carrier or excipient.

Thus, included are pharmaceutical compositions comprising apharmaceutically acceptable carrier or excipient and at least onecompound of formula

wherein R₁-R₄ are independently selected from the group consisting of:

(a) a straight chain or branched lower alkyl group substituted with aheterologous nitrogen-containing ring group of five to seven members;

(b) in combination with the O attached to the phenyl ring, a carbamategroup, other than a methyl carbamate group, optionally containing a fiveor six membered carbon-nitrogen ring;

(c) in combination with the O attached to the phenyl ring, a C₁-C₆straight chain or branched carbonate group; and

(d) wherein R₁ and R₂; or R₃ and R₄; or R₁ and R₂, and R₃ and R₄;together with the phenyl group to which they are attached, combine toform a cyclic carbonate group;

or a salt of the compound.

The above-mentioned compositions are used in a method of treatment of adisease or disorder in a subject comprising administering to the subjectan effective amount of the composition to treat the disease or disorder.Diseases and disorders to be treated include, inter alia, viral andproliferative diseases (e.g. cancer), as mentioned in detail below.

According to the methods described herein, the compounds andpharmaceutical compositions are administered to an individual in need oftreatment, especially a mammal (e.g. a human), in amounts effective fortreating a disease or disorder with which the individual is afflicted.

The above-mentioned compounds are also useful in methods of inhibitingexpression of a Sp1-regulated eukaryotic gene in a mammalian cell orsubject, of inhibiting HIV Tat-regulated transactivation in a cell orsubject, and of widening the major groove of a double strandeddeoxyoligonucleotide containing an Sp1 binding site ‘5GGGCGGG3’(dsOLIGO_(sp)). As such, they will be useful in methods for treatingdiseases and disorders involving these processes, as described infurther detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Stability of Two NDGA Derivatives G₄N and P₄N(HCl salts) inAqueous Solution. Time Dependent Changes in UV spectra showing (a) G₄Nis unstable and (b)meso-2,3-dimethyl-1,4-bis(3,4-[2-piperidino)ethoxyphenyl])butane (P₄N)(compound 5b in Scheme 1) (day 1, 2, 8, 13, 28) is stable in Milli-Qwater after 28 days at 20° C.

FIG. 2: Major groove of DNA widens in the presence of P₄N. Space fillingrepresentations of the crystal structure of a 12mer crystallized withSpermidine (12mer-spd, with carbon atoms colored green) and of a 12mer,dsOLIGOsp (dGGGGCGGGG with two or three additional base-pairs on eitherthe 5′ or 3′ ends) crystallized in the presence of spermidine and P₄N(12mer-spd-P₄N, with carbon atoms colored yellow) illustrating thewidening of the major groove of DNA in the presence of P₄N (Ref. 25, JMB349, 731-744 2005).

FIG. 3: 1D 1H NMR spectra of free DNA, free P4N and 1:1 molar ratioDNA:P4N. The resonances with the largest chemical shift perturbation areindicated

dsOLIGOsp. 5′GATGGGCGGGACG3′ 3′CTACCCGCCCTGC5

FIG. 4: Superposition of representative region of the 2D 1H-13C HSQCspectra of the free DNA (green) free P4N (red) and 1:1 complex (blue).The spectra were collected with folding in 13C dimension for improvedresolution.

FIG. 5: First derivate (dA/DT) of the absorbance (A) with respect toTemperature (T) as a function of temperature.

FIG. 6: Electrophoretic Mobility-Shift Analysis of Interaction of HIVLTR(−87 to −49) with Sp1 Protein in the presence and absence of competitorP₄N. Blocking the Sp1 binding to (A) and replacement of the bound Sp1from (B). HIVLTR (−87 to −49) by P₄N: 2 ng ³²P-labeled HIVLTR, wasincubated with P₄N (0, 50, 100, 200, 400, 800 μM) for 30 minutes.Reaction was continued for additional 30 minutes in the presence of 2 ngof recombinant Sp1 −167C(A). 2 ng of ³²P-labeled HIVLTR was incubatedwith 2 ng of recombinant Sp1 −167C for 30 minutes. Reaction wascontinued for additional 30 minutes in the presence of a series ofconcentrations of P₄N(HCl salt) (0, 50, 100, 200, 400, 800 μM) (B).(Reference 10.)

FIG. 7: Consistent Valence Forcefilled (EVFF) Calculations of P₄N in theMajor Groove of dsOLIGOMERsp.

FIG. 8: Tat Induced HIV Transactivation. SeAP standard assay forsecreted alkaline phosphatase (Reference 21) Induction of HIVLTRpromoter activity by HIV Tat (Reference 12). SeAP level (A₄₀₅) isplotted against time (minutes).

FIG. 9: Inhibition of HIV Tat-Regulated transactivation by Compound 5bP₄N. The COS cells were co-transfected with a Tat-producing plasmid anda plasmid containing the HIV LTR linked to the SeAP reporter gene.Transfected cells were treated with various concentrations of 5b P₄N.After 48 h, aliquots of media were removed and analysis of SeAP activitywas performed. The percent inhibition of SeAP activity by 5b P₄N

was calculated in comparison with the percent of growth inhibition ofuntransfected COS cells by P₄N

FIG. 10: Efficacy of P₄N Against HSV-2 in a Mouse Vaginal Model

FIG. 11: Effect of P₄N on Proliferation of VERO Cells and Three HumanCancer Cells in Culture.

DETAILED DESCRIPTION

The invention described below is given by way of example only and is notto be interpreted in any way as limiting the invention.

The present inventors have surprisingly discovered that the compoundsand compositions containing the NDGA derivatives described herein areparticularly effective for the treatment of viral infection andproliferative diseases such as tumors. In addition to the properties ofimproved water solubility (Scheme 1 compounds) and stability, thecompounds should be suitable for systemic treatment. Although notwishing to be bound by any particular theory, it is believed that thesecompounds are advantageous in that they exert their pharmaceuticaleffects by binding to the Sp1 binding site in a reversible manner, thuscausing less systemic toxicity, while still exhibiting good efficacyagainst the targeted disease or disorder.

Definitions

As used herein, the term “lower alkyl” means C₁-C₆ alkyl.

As used herein, the term “lower alkyoxy” means C₁-C₆ alkoxy.

As used herein, “heterologous ring” means a 5-7 membered carbon-nitrogenor carbon-oxygen ring.

As used herein, the term “NDGA derivative” refers to a derivative ofNDGA having the formula:

wherein

wherein R₁-R₄ are independently selected from the group consisting of:

(a) a straight chain or branched lower alkyl group substituted with aheterologous nitrogen-containing ring group of five to seven memberssuch as a pyrrolidino, piperidino, or morpholino group;

(b) in combination with the O attached to the phenyl ring, a carbamategroup, other than a methyl carbamate group, optionally containing a fiveor six membered carbon-nitrogen ring;

(c) in combination with the O attached to the phenyl ring, a C₁-C₆straight chain or branched carbonate group; and

(d) wherein R₁ and R₂; or R₃ and R₄; or R₁ and R₂, and R₃ and R₄;together with the phenyl group to which they are attached, combine toform a cyclic carbonate group;

or a salt of the compound.

By “carbamate” is meant a substituent, in combination with the Oattached to the phenyl ring, of the group —O—C(O)—NHR, or —O—C(O—NR₅R₆,wherein R, R₅ or R₆ represent any carbon-containing group as furtherdefined herein, or wherein R₅ and R₆ together complete anitrogen-containing ring.

By “carbonate” is meant a substituent, in combination with the 0attached to the phenyl ring, of the group —O—CO—OR, wherein R representsany carbon-containing group as further defined herein.

As used herein, “buffers” includes any buffer conventional in the art,such as, for example, Tris, phosphate, imidazole, and bicarbonate.

As used herein, “target tissue” means any tissue to which it is desiredto deliver an effective concentration of a compound of the invention,e.g., blood, brain, a specific tumor.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a condition or disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a condition ordisease and/or adverse affect attributable to the condition or disease.“Treatment,” thus, for example, covers any treatment of a condition ordisease in a mammal, particularly in a human, and includes: (a)preventing the condition or disease from occurring in a subject whichmay be predisposed to the condition or disease but has not yet beendiagnosed as having it; (b) inhibiting the condition or disease, suchas, arresting its development; and (c) relieving, alleviating orameliorating the condition or disease, such as, for example, causingregression of the condition or disease.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any conventional type. A “pharmaceuticallyacceptable carrier” is non-toxic to recipients at the dosages andconcentrations employed, and is compatible with other ingredients of theformulation. For example, the carrier for a formulation containing thepresent compounds preferably does not include oxidizing agents and othercompounds that are known to be deleterious to such. Suitable carriersinclude, but are not limited to, water, dextrose, glycerol, saline,ethanol, buffer, dimethyl sulfoxide, Cremaphor EL, and combinationsthereof. The carrier may contain additional agents such as wetting oremulsifying agents, or pH buffering agents. Other materials such asanti-oxidants, humectants, viscosity stabilizers, and similar agents maybe added as necessary.

Pharmaceutically acceptable salts herein include the acid addition saltswhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, mandelic, oxalic,and tartaric. Salts may also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, and histidine.

The term “pharmaceutically acceptable excipient,” includes vehicles,adjuvants, or diluents or other auxiliary substances, such as thoseconventional in the art, which are readily available to the public. Forexample, pharmaceutically acceptable auxiliary substances include pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents and the like.

The terms “individual”, “subject,” “host,” and “patient,” are usedinterchangeably herein to refer to an animal being treated with thepresent compositions, including, but not limited to, simians, humans,felines, canines, equines, bovines, porcines, ovines, caprines,mammalian farm animals, mammalian sport animals, and mammalian pets.

As used herein “parenteral administration” herein means intravenous,intra-arterial, intramuscular, subcutaneous, transdermal, intradermaland intraperitoneal administration.

A “substantially purified” compound in reference to the compoundsdescribed herein is one that is substantially free of compounds that arenot the compound in question. By “substantially free” is meant at least50%, preferably at least 70%, more preferably at least 80%, and evenmore preferably at least 90%, most preferably greater than 95% or 99%free of extraneous materials.

It is to be understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a compound” includes a plurality of suchcompounds and equivalents thereof known to those skilled in the art.

Compounds disclosed herein may be made by the methods described herein,or by any other methods that will be known to those of skill in the art.

Overview of Synthesis of NDGA Derivatives.

Exemplary compounds of the present invention were prepared by treatingNDGA (1) with an alkyl chloride bearing a hydrocarbon spacer and anitrogen-containing five- or six-membered ring in the presence of sodiumcarbonate and acetone (see Scheme 1). These intermediates were thenallowed to react with HCl_((g)) in situ to give tetra-O-alkylated NDGA5a-d in 67-82% overall yields. Their solubility in aqueous solution wasfound 379-541 mg/mL.

Several new lipophilic NDGA derivatives in the families of carbamate andcarbonate were prepared as shown in Scheme 2. Treatment of NDGA with1-piperidinecarbonyl chloride in the presence of pyridine at 0° C.produced the NDGA carbamate 6a in 72% yield. Under the same conditions,NDGA reacted with methyl chloroformate or 1,1′-carbonyldiimidazoleafforded carbonates 6b and 7, respectively. The latter product wasgenerated through an intramolecular cyclization process.

The new NDGA derivatives (Scheme 1) were found stable in aqueoussolution; >99% of these compounds remained intact after 28 days (seeFIG. 1( b)). In a sharp contrast, >96% G₄N (4) decomposed in aqueoussolution within 24 h (see FIG. 1( a)).

General Procedure.

All reactions were carried out in oven-dried glassware (120° C.) underan atmosphere of nitrogen, unless as indicated otherwise. Acetone,dichloromethane, 1,4-dioxane, ethyl acetate, hexane, and tetrahydrofuranwere purchased Mallinckrodt Chemical Co. Acetone was dried with 4 Åmolecular sieves and distilled. Dichloromethane, ethyl acetate, andhexane were dried and distilled from CaH₂. 1,4-Dioxane andtetrahydrofuran were dried by distillation from sodium and benzophenoneunder an atmosphere of nitrogen. Nordihydroguaiaretic acid was purchasedfrom Fluka Chemical Co. 4-(2-Chloroethyl)morpholine hydrochloride,1-(2-chloroethyl)piperidine monohydrochloride,1-(2-chloroethyl)pyrrolidine hydrochloride,N,N′-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), andpotassium carbonate were purchased from Aldrich Chemical Co.

The melting point was obtained with a Buchi 535 melting point apparatus.Analytical thin layer chromatography (TLC) was performed on precoatedplates (silica gel 60 F-254), purchased from Merck Inc. Gaschromatographic analyses were performed on a Hewlett-Packard 5890 SeriesII instrument equipped with a 25-m crosslinked methyl silicone gumcapillary column (0.32 mm i.d.). Nitrogen gas was used as a carrier gasand the flow rate was kept constant at 14.0 mL/min. The retention timet_(R) was measured under the following conditions: injector temperature260° C., isothermal column temperature 280° C. Gas chromatography andlow resolution mass spectral analyses were performed on a AgilentTechnology 6890N Network GC System equipped with a Agilent 5973 NetworkMass Selective Detector and capillary HP-1 column. Purification bygravity column chromatography was carried out by use of Merck ReagentsSilica Gel 60 (particle size 0.063-0.200 mm, 70-230 mesh ASTM). Purityof all compounds was >99.5%, as checked by HPLC or GC.

Ultraviolet (UV) spectra were measured on a Hitachi U3300 UV/VISspectrophotometer. Infrared (IR) spectra were measured on a JascoFT-IR-5300 Fourier transform infrared spectrometer. The wave numbersreported were referenced to the polystyrene 1601 cm⁻¹ absorption.Absorption intensities were recorded by the following abbreviations: s,strong; m, medium; w, weak. The fluorescent intensity was measured on aHitach F-4500 Florescence Spectrophotometer. Proton NMR spectra wereobtained on a Varian Mercury-400 (400 MHz) spectrometer by use ofchloroform-d as the solvent and sodium 3-(trimethylsilyl)propionate asinternal standard. Carbon-13 NMR spectra were obtained on a VarianMercury-400 (100 MHz) spectrometer by use of chloroform-d or D₂O as thesolvent. Carbon-13 chemical shifts were referenced to the center of theCDCl₃ triplet (δ77.0 ppm). Multiplicities are recorded by the followingabbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m,multiplet; J, coupling constant (hertz). High-resolution mass spectrawere obtained by means of a JEOL JMS-HX110 mass spectrometer.Electrospray ionization mass spectrometry (ESI-MS) analyses wereperformed on a quadrupole ion trap mass analyzer fitted with anelectrospray ionization source of Finnigan LCQ, Finnigan MAT.

Computation was performed on a Silicon Graphics O2+ workstation. Theprograms Builder and Biopolymer were used for the construction ofstructures. The program Discover was used for energy minimized with theconsistent valence force field (CVFF) until the maximum derivative wasless than 1.0 kcal mol⁻¹ Å⁻¹.

Standard Procedure for the Syntheses of Hydrochloride Salts of NDGADerivatives.

To a solution containing NDGA (1, 1.0 equiv) and potassium carbonate(6.0-10.0 equiv) in acetone was added a nitrogen-containing organichydrochloride (5.0 equiv). After the solution was heated at reflux for24 h, it was quenched with water (20 mL). The solution was extractedwith ether (3×50 mL) and the combined organic layers were washed withsaturated brine, dried over MgSO_(4(s)), filtered, and concentratedunder reduced pressure. The residue was purified by columnchromatography on silica gel (10% methanol in dichloromethane as eluant)and the desired fraction was concentrated. The resultant was dissolvedin acetone (250 mL) and then bubbled with excess HCl_((g)). Theprecipitates were dissolved in water and re-precipitated twice by use ofacetone at room temperature to give the desired NDGA derivative withpurity >99.5%, as checked by HPLC. This procedure was varied asdescribed below for individual NDGA derivatives.

Details of the preparation of specific NDGA derivative compoundsaccording to the present invention will be set forth below in theExamples section.

The present compounds and compositions, in suitable formulations, can besafely administered to a subject in need of such treatment by anyeffective route known in the art. Such means of administration are knownin the art and can be determined by routine experimentation. Examplesare intranasal administration; oral administration; inhalationadministration; subcutaneous administration; transdermal administration;intradermal administration; intra-arterial administration, with orwithout occlusion; intracranial administration; intraventricularadministration; intravenous administration; buccal administration;intraperitoneal administration; intraocular administration;intramuscular administration; implantation administration; topicaladministration; and central venous administration. Additionally, thecompounds and compositions can be administered as an oral rinse, forexample, in a rinse-and-spit treatment one or more times a day.

Moreover, the compounds and compositions can be formulated in liposomalformulations, nanoparticle formulations, or micellar formulations thatcan be safely administered systemically, such as intravenously, such asby injection into the central vein for example, or intraperitoneally,interstitially, subcutaneously, transdermally, intradermally,intramuscularly, intra-arterially, intra-cranially, orintra-ventricularly.

Furthermore, the compounds and compositions can be formulated inliposomal formulations, nanoparticles formulations, or micellarformulations, or any formulation embedded in a biodegradable polymer,for administration to a subject. Implantation into the brain, forexample, can be used for treatment of brain tumors.

The pharmaceutical compositions provided herein are formulated fordelivery or administration as described above, for example, in the formof a tablet, a liquid that is either hydrophilic or hydrophobic, apowder such as one resulting from lyophilization, an aerosol, or in theform of an aqueous water-soluble composition, a hydrophobic composition,a liposomal composition, a micellar composition, such as that based onTween® 80 or diblock copolymers, a nanoparticle composition, a polymercomposition, a cyclodextrin complex composition, emulsions, lipid basednanoparticles termed “lipocores.” In one preferred embodiment, thepharmaceutical composition comprises one or more compounds of theinvention in aqueous solution.

The present invention further features a method of producing thepharmaceutical compositions of the present invention, the methodinvolving making or providing a compound of the invention in asubstantially purified form, combining the composition with apharmaceutically acceptable carrier or excipient, and formulating thecomposition in a manner that is compatible with the mode of desiredadministration.

In a further aspect of the present invention, a method of treating aproliferative disease, including pre-malignant, benign or malignantcancer, is provided. Non-limiting examples of cancers and tumors includethose of the lung, prostate, breast, colon, liver, kidney, ovarian,cervical, skin, pancreas, brain, nasal, pharyngeal, head, neck,leukemias, lymphomas, gastrointestinal tumor such as stomach andbladder, soft tissue sarcomas and the like, as well as metastasesthereof.

The present invention still additionally provides for kits comprisingcompounds or compositions as above for the treatment of diseases anddisorders, in particular viral infections or proliferative diseases suchas tumors, wherein the compositions are formulated for delivery asabove, including but not limited to intranasal administration,inhalation, oral administration, intravenous administration,intraperitoneal administration and other parenteral administration, oras an oral rinse, or the like. Accordingly, the kits comprise apharmaceutical composition of the invention in a suitable dosage, andoptionally instructions for administration of the composition.

Pharmaceutical compositions provided herein include one or more of thecompounds mentioned above and at least one pharmaceutically acceptablecarrier or excipient. These compositions may include a buffer, which isselected according to the desired use of the composition, and may alsoinclude other substances appropriate for the intended use. Those skilledin the art can readily select an appropriate buffer, a wide variety ofwhich are known in the art, suitable for an intended use. In someinstances, the composition can comprise a pharmaceutically acceptableexcipient, a variety of which are known in the art. Pharmaceuticallyacceptable excipients suitable for use herein are described in a varietyof publications, including, for example, A. Gennaro (1995 Gennaro, A.(1995). “Remington: The Science and Practice of Pharmacy”, 19th edition,Lippincott, Williams, & Wilkins; Ansel, H. C. et al. (1999),Pharmaceutical Dosage Forms and Drug Delivery Systems eds., 7^(th) ed.,Lippincott, Williams, & Wilkins; Kibbe, A. H. (2000) Handbook ofPharmaceutical Excipients, eds., 3^(rd) ed. Amer. Pharmaceutical Assoc.

The compositions herein are formulated in accordance to the mode ofpotential administration. Thus, if the composition is intended to beadministered intranasally or by inhalation, for example, the compositionmay be converted to a powder or aerosol form, as conventional in theart, for such purposes. Other formulations, such as for oral orparenteral delivery, are also used as conventional in the art.

Compositions for administration herein may form solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders.

Therapeutic Methods

The compounds and compositions of the subject invention find use astherapeutic agents in situations, for example, where one wishes toprovide a treatment to a subject who has a proliferative disease such asa malignant, premalignant or benign tumor and where one wishes toprovide treatment to viral diseases such as HIV, HPV or HSV.

A variety of animal hosts are treatable according to the subjectmethods, including human and non-human animals. Generally such hosts are“mammals” or “mammalian,” where these terms are used broadly to describeorganisms which are within the class mammalia, including the orderscarnivore (e.g., dogs and cats), rodentia (e.g., guinea pigs, and rats),and other mammals, including cattle, goats, horses, sheep, rabbits,pigs, and primates (e.g., humans, chimpanzees, and monkeys). In manyembodiments, the hosts will be humans. Animal models are of interest forexperimental investigations, such as providing a model for treatment ofhuman disease. Further, the present invention is applicable toveterinary care as well.

The compounds and compositions of the present invention can be used totreat a variety of tumors and cancers, including, without limitation,hematological malignancies such as acute lymphoblastic leukemia, acutemyeloid leukemia, chronic lymphocytic leukemia, chronic myelogenousleukemia, childhood acute leukaemia, non-Hodgkin's lymphoma, chroniclymphocytic leukaemia, malignant cutaneous T-cells, mycosis fungoides,non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell richcutaneous lymphoid hyperplasia, bullous pemphigoid, discoid lupuserythematosus, lichen planus, adrenocortical carcinoma, anal cancer,astrocytoma, bile duct cancer, bladder cancer, bone cancerosteosarcoma/malignant fibrous histiocytoma, neurological malignanciessuch as neuroblastoma, glioblastoma, astrocytoma, gliomas, brain stemglioma, brain tumor ependymoma, brain tumor medulloblastoma,neuroblastoma glioblastoma, breast cancer, carcinoid tumorgastrointestinal, carcinoma adrenocortical, carcinoma islet cell,cervical cancer, clear cell sarcoma of tendon sheaths, colon cancer,cutaneous T-cell lymphoma, endometrial cancer, epithelial cancerovarian, esophageal cancer, Ewing's family of tumors, extragonadal germcell tumor, extrahepatic bile duct cancer, eye cancer, intraocularmelanoma, ductal cancer, eye cancer retinoblastoma, dysplastic oralmucosa, invasive oral tumor, gallbladder cancer, gastric (stomach)cancer, gastrointestinal carcinoid tumor, germ cell tumor extragonadal,germ cell tumor, ovarian tumor, gestational trophoblastic tumor,glioblastoma, glioma, hairy cell leukemia, hepatocellular (liver)cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma,islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, laryngealcancer, leukemia acute lymphoblastic cancer, leukemia acute myeloidcancer, leukemia chronic lymphocytic cancer, leukemia chronicmyelogenous cancer, leukemia hairy cell cancer, liver cancer, non-smallcell lung cancer, small cell lung cancer, male breast cancer, malignantmesothelioma, medulloblastoma, melanoma, merkel cell carcinoma, multipleendocrine neoplasia syndrome, mycosis fungoides, myeloma multiple, nasalcavity, paranasal and sinus cancer, nasopharyngeal cancer,neuroblastoma, oral cavity and lip cancer, oropharyngeal cancer,osteosarcoma/malignant fibrous histiocytoma of bone, ovarian epithelialcancer, ovarian germ cell tumor, pancreatic cancer, parathyroid cancer,penile cancer, pheochromocytoma, pineal and supratentorial primitiveneuroectodermal tumors, pituitary tumor, pleuropulmonary blastoma,prostate cancer, rectal cancer, renal, pelvis and ureter transitionalcell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer,sarcoma soft tissue adult, Sezary syndrome, skin cancer, small intestinecancer, stomach (gastric) cancer, testicular cancer, thymoma, thyroidcancer, urethral cancer, transitional and squamous cell urinarycarcinoma, gynecological cancer such as cervical cancer ovarian cancer,uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer,Waldenström's macroglobulinemia, and Wilms' tumor, testicular tumors;liver tumors including hepatocellular carcinoma (“HCC”) and tumor of thebiliary duct; multiple myelomas; tumors of the esophageal tract; otherlung tumors including small cell and clear cell; Hodgkin's lymphomas;sarcomas in different organs; as well as those mentioned above; and thelike

Formulations, Dosages, and Routes of Administration

As mentioned above, an effective amount of the active agent isadministered to the host, where “effective amount” means a dosagesufficient to produce a desired result. In some embodiments, the desiredresult is at least a reduction or inhibition of tumor growth as comparedto a control.

Typically, the compositions of the instant invention will contain fromless than about 1% up to about 99% of the active ingredient, that is,the compounds described herein, preferably with a pharmaceuticallyacceptable carrier or excipient; optionally, the instant invention willcontain about 5% to about 90% of the active ingredient. The appropriatedose to be administered depends on the subject to be treated, such asthe general health of the subject, the age of the subject, the state ofthe disease or condition, the weight of the subject, the size of thetumor, for example. Generally, between about 0.1 mg and about 500 mg orless may be administered to a child and between about 0.1 mg and about 5grams or less may be administered to an adult. The active agent can beadministered in a single or, more typically, multiple doses. Preferreddosages for a given agent are readily determinable by those of skill inthe art by a variety of means. Other effective dosages can be readilydetermined by one of ordinary skill in the art through routine trialsestablishing dose response curves. The amount of agent will, of course,vary depending upon the particular agent used.

In an alternative embodiment, dosage is defined in terms of the amountof agent delivered to a target tissue (e.g. a tumor, or an organ). Inthis instance, dosages may be defined as concentrations, e.g. 0.01 μM-10mM, 0.01 μM-100 mM, etc.

The frequency of administration of the active agent, as with the doses,will be determined by the practitioner based on age, weight, diseasestatus, health status and patient responsiveness. Thus, the agents maybe administered one or more times daily, weekly, monthly or asappropriate as conventionally determined. The agents may be administeredintermittently, such as for a period of days, weeks or months, then notagain until some time has passed, such as 3 or 6 months, and thenadministered again for a period of days, weeks, or months.

The compounds of the present invention can be incorporated into avariety of formulations for therapeutic administration. Moreparticularly, the compounds can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, aerosols, liposomes, nanoparticles, granules,ointments, solutions, suppositories, injections, inhalants and aerosols.

As such, administration of the compounds can be achieved in variousways, such as oral, buccal, rectal, intranasal, intravenous,intra-arterial, intra-tracheal, intraventricular, intracranial,interstitial, transdermal, etc., or by inhalation or implantation.

In particular, nanoparticle, micelle and liposomal preparation can beadministered systemically, including parenterally and intranasally, aswell as interstitially, orally, topically, transdermally, intradermally,via inhalation or implantation, such as for drug targeting, enhancementof drug bioavailability and protection of drug bioactivity andstability. Nanoparticle bound drugs herein are expected to achieveprolonged drug retention in tumors.

In pharmaceutical dosage forms, the compounds may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the compounds can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents. For oral rinses,the preparations can be made in a manner conventional in the art, suchas described in, for example, Epstein, J. B. et al. (2002). Fluconazolemouthrinses for oral candidiasis in post-irradiation, transplant, andother patients. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod.93(6): 671-675. and Pitten, F. et al. (2003) Do cancer patients withchemotherapy-induced leucopenia benefit from an antisepticchlorhexidine-based oral rinse? A double-blind, block-randomized,controlled study. J. Hosp. Infect. 53(4): 283-291.

Pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are conventional in the art. Suitable excipientvehicles are, for example, water, saline, dextrose, glycerol, ethanol,or the like, and combinations thereof. In addition, if desired, thevehicle may contain minor amounts of auxiliary substances such as pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents or emulsifying agents. Actual methods of preparing suchdosage forms are known, or will be apparent, to those skilled in theart. See, e.g., Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., 17th edition, 1985. The composition or formulationto be administered will, in any event, contain a quantity of the agentadequate to achieve the desired state in the subject being treated.

The active agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or non-aqueoussolvent, such as vegetable or other similar oils, including corn oil,castor oil, synthetic aliphatic acid glycerides, esters of higheraliphatic acids or propylene glycol; and if desired, with conventionaladditives such as solubilizers, isotonic agents, suspending agents,emulsifying agents, stabilizers and preservatives.

The active agents can be utilized in aerosol formulation to beadministered via inhalation. The compounds of the present invention canbe formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the active agents can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. The compounds of the present invention can be administeredrectally via a suppository. The suppository can include vehicles such ascocoa butter, carbowaxes and polyethylene glycols, which melt at bodytemperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

Kits with multiple or unit doses of the active agent, are included inthe present invention. In such kits, in addition to the containerscontaining the multiple or unit doses of the compositions containing theNDGA derivatives, an informational package insert with instructionsdescribing the use and attendant benefits of the drugs in treatingpathological condition of interest may optionally be included.

Preparation of NanoParticles (“NP”)

The present invention includes formulations of the compounds of theinvention in a NP preparation. A number of different NP formulationssuitable for use herein can be made depending on the method of delivery.The NP formulation can differ based on the drug release profile desired,by controlling the molecular weight, the copolymer ratio, the drugloading, the microparticle size and porosity and the fabricationconditions. The NP formulations can also differ on the basis ofpolymers, stabilizers, and surfactants used in the production process.Different excipients may also have different effects on drug uptake,drug distribution throughout the body and persistence of the drug inplasma. A person having skills conventional in the art will be able todetermine the desired properties or characteristics, and accordinglydetermine the appropriate NP formulation to use.

The polymeric matrix of the NP must meet the criteria ofbiocompatibility, bioavailability, mechanical strength and ease ofprocessing. The best known polymers for this purpose is thebiodegradable poly(lactide-co-glycolide)s (“PLGAs”).

NP for use in the pharmaceutical preparations can be made by any processconventional in the art. In one embodiment, the NP can be made asdescribed in, for example, Lockman, P. R., et al. (2002), NanoparticleTechnology for Drug Delivery Across the Blood-Brain Barrier. DrugDevelopment Indus. Pharmacy, 28(1): 1-13. The types of manufacturingprocess include, for example, emulsion polymerization, interfacialpolymerization, desolvation evaporation and solvent deposition.

In the emulsion polymerization process of making the NP herein, thepolymerization process consists of building a chain of polymers from asingle monomer unit, as described in, for example, Kreuter, J. (1994),Nanoparticles, In Encyclopedia of Pharmaceutical Technology; Swarbick,J.; Boylan, J. C. Eds.; Marcel Dekker (New York, 1994), pp. 165-190.Polymerization occurs spontaneously at room temperature after initiationby either free radical or ion formation, such as by use of high-energyradiation, UV light, or hydroxyl ions. Once polymerization is completethe solution is filtered and neutralized. The polymers form micelles anddroplets consisting of from about 100 to 10⁷ polymer molecules.Surfactants and stabilizers are generally not need in this process. Thisprocess can also be accomplished in an organic phase rather than anaqueous phase.

The NP herein can also be made by an interfacial polymerization processas described in, for example, Khouri, A. I., et al. (1986), Developmentof a new process for the manufacture of polyisobutyl-cyanoacrylatenanoparticles, Int. J. Pharm., 28: 125. In this process, monomers areused to create the polymer and polymerization occurs when an aqueous andorganic phase are brought together by homogenization, emulsification, ormicro-fluidization under high-torque mechanical stirring. For example,polyalkylcyanoacrylate nanocapsules containing the catecholic butanes,such as the NDGA compounds of this invention, can be made by combiningthe lipophilic NDGA compounds and the monomer in an organic phase,dissolving the combination in oil, and slowly adding the mixture througha small tube to an aqueous phase with constant stirring. The monomer canthen spontaneously form 200-300 nm capsules by anionic polymerization. Avariation of this process involves adding a solvent mixture of benzylbenzoate, acetone, and phospholipids to the organic phase containing themonomer and the drug, as described in, for example, Fessi, H., et al.(1989). Nanocapsule formulation by interfacial deposition followingsolvent displacement. Int. J. Pharm., 55: R1-R4. This creates aformulation in which the drug is encapsulated and protected againstdegradation until it reaches the target tissue.

Macromolecules such as albumin and gelatin can be used in oildenaturation and desolvation processes in the production of NPs. In theoil emulsion denaturation process, large macromolecules are trapped inan organic phase by homogenization. Once trapped, the macromolecule isslowly introduced to an aqueous phase undergoing constant stirring. Thenanoparticles formed by the introduction of the two immiscible phasescan then be hardened by crosslinking, such as with an aldehyde or byheat denaturation.

Alternatively, macromolecules can form NPs by “desolvation.” In thedesolvation process, macromolecules are dissolved in a solvent in whichthe macromolecules reside in a swollen, coiled configuration. Theswollen macromolecule is then induced to coil tightly by changing theenvironment, such as pH, charge, or by use of a desolvating agent suchas ethanol. The macromolecule may then be fixed and hardened bycrosslinking to an aldehyde. The NDGA compounds can be adsorbed or boundto the macromolecule before crosslinking such that the derivativesbecome entrapped in the newly formed particle.

Solid lipid NP can be created by high-pressure homogenization. Solidlipid NPs have the advantage that they can be sterilized and autoclavedand possess a solid matrix that provides a controlled release.

The present invention further includes NP with different methods of drugloading. The NP can be solid colloidal NP with homogeneous dispersion ofthe drug therein. The NP can be solid NP with the drug associated on theexterior of the NP, such as by adsorption. The NP can be a nanocapsulewith the drug entrapped therein. The NP can further be solid colloidalNP with homogeneous dispersion of the drug therein together with a cellsurface ligand for targeting delivery to the appropriate tissue.

The size of the NPs may be relevant to their effectiveness for a givenmode of delivery. The NPs typically ranges from about 10 nm to about1000 nm; optionally, the NPs can range from about 30 to about 800 nm;further typically, from about 60 to about 270 nm; even furthertypically, from about 80 to about 260 nm; or from about 90 to about 230nm, or from about 100 to about 195. Several factors influence the sizeof the NPs, all of which can be adjusted by a person of ordinary skillin the art, such as, for example, pH of the solution used duringpolymerization, amount of initiation triggers (such as heat orradiation, etc.) and the concentration of the monomer unit. Sizing ofthe NPs can be performed by photon correlation spectroscopy using lightscattering.

The NPs herein, such as polysaccharide NPs or albumin NPs, mayoptionally be coated with a lipid coating. For example, polysaccharideNPs can be crosslinked with phosphate (anionic) and quarternary ammonium(cationic) ligands, with or without a lipid bilayer, such as onecontaining dipalmitoyl phosphatidyl choline and cholesterol coating.Other polymer/stabilizer include, but is not limited to: soybean oil;maltodextrin; polybutylcyanoacrylate; butylcayanoacrylate/dextran 70kDa, Polysorbate-85; polybutylcyanoacrylate/dextran 70 kDa,polysorbate-85; stearic acid; poly-methylmethylacrylate.

The NP preparations containing the compounds of the invention, such asby adsorption to the NPs, can be administered intravenously fortreatment of tumors, for example, in the brain, heart andreticuloendothelial cell (“RES”) containing organs, such as liver,spleen and bone marrow. To avoid undesirable uptake of these NPpreparations by the reticuloendothelial cells, the NPs may be coatedwith a surfactant or manufactured with a magnetically responsivematerial.

Thus, optionally, a surfactant may be used in conjunction with the NP.For example, polybutylcyanoacrylate NPs can be used with adextran-70,000 stabilizer and Polysorbate-80 as a surfactant. Othersurfactants include, but not limited to: Polysorbate-20, 40, or 60;Poloxamer 188; lipid coating-dipalmitoyl phosphatidylcholine; Epikuron200; Poloxamer 338; Polaxamine 908; Polaxamer 407. For example,Polyaxamine 908 may be used as a surfactant to decrease uptake of NPsinto the RES of the liver, spleen, lungs, and bone marrow.

The magnetically responsive material can be magnetite (Fe₃O₄) which canbe incorporated into the composition for making the NP. Thesemagnetically responsive NPs can be externally guided by a magnet.

In another embodiment, the NPs herein can be made as described in Mu, L.and Feng, S. S. (2003) (A novel controlled release formulation for theanticancer drug paclitaxel (Taxol®): PLGA nanoparticles containingvitamin E TPGS. J. Control. Rel. 86: 33-48), using a blend ofpoly(lactide-co-glycolide)s (“PLGAs”) and d-a-tocopheryl polyethyleneglycol 1000 succinate (vitamin E TPGS or TPGS). The latter can also actas an emulsifier, in addition to being a matrix material.

Preparation of Micelle Forming Carriers

The present invention includes the disclosed NDGA derivatives,formulated in micelle forming carriers, where the micelles are producedby processes conventional in the art. Examples of such are described in,for example, Liggins, R. T. and Burt, H. M. (2002) Polyether-polyesterdiblock copolymers for the preparation of paclitaxel loaded polymericmicelle formulations. Adv. Drug Del. Rev. 54: 191-202; Zhang, X. et al.(1996) Development of amphiphilic diblock copolymers as micellarcarriers of taxol., Int. J. Pharm. 132: 195-206; and Churchill, J. R.,and Hutchinson, F. G. (1988). Biodegradable amphipathic copolymers. U.S.Pat. No. 4,745,160. In one such method, polyether-polyester blockcopolymers, which are amphipathic polymers having hydrophilic(polyether) and hydrophobic (polyester) segments, are used as micelleforming carriers.

Another type of micelles is, for example, that formed by the AB-typeblock copolymers having both hydrophilic and hydrophobic segments, whichare known to form micellar structures in aqueous media due to theiramphiphilic character, as described in, for example, Tuzar, Z. andKratochvil, P. (1976). Block and graft copolymer micelles in solution.Adv. Colloid Interface Sci. 6:201-232; and Wilhelm, M. et al. (1991).Poly(styrene-ethylene oxide) block copolymer micelle formation in water:a fluorescence probe study. Macromolecules 24: 1033-1040. Thesepolymeric micelles are able to maintain satisfactory aqueous stabilityirrespective of the high content of hydrophobic drug incorporated withinthe micelle inner core. These micelles, in the range of approximately<200 nm in size, are effective in reducing non-selective RES scavengingand shows enhanced permeability and retention at solid tumor sites. Thischaracteristic allows for the accumulation of anti-cancer drug, such asthe NDGA derivatives, to accumulate at the cancer site.

Further, for example, poly(D,L-lactide)-b-methoxypolyethylene glycol(MePEG:PDLLA) diblock copolymers can be made using MePEG 1900 and 5000.The reaction can be allowed to proceed for 3 hr at 160° C., usingstannous octoate (0.25%) as a catalyst. However, a temperature as low as130° C. can be used if the reaction is allowed to proceed for about 6hr, or a temperature as high as 190° C. can be used if the reaction iscarried out for only about 2 hr.

In one embodiment, N-isopropylacrylamide (“IPAAm”) (Kohjin, Tokyo,Japan) and dimethylacrylamide (“DMAAm”) (Wako Pure Chemicals, Tokyo,Japan) can be used to make hydroxyl-terminated poly(IPAAm-co-DMAAm) in aradical polymerization process, using the method of Kohori, F., et al.(1998). Preparation and characterization of thermally responsive blockcopolymer micelles comprising poly(N-isopropylacrylamide-b-D,L-lactide).J. Control. Rel. 55: 87-98. The obtained copolymer can be dissolved incold water and filtered through two ultrafiltration membranes with a10,000 and 20,000 molecular weight cut-off. The polymer solution isfirst filtered through a 20,000 molecular weight cut-off membrane. Thenthe filtrate was filtered again through a 10,000 molecular weightcut-off membrane. Three molecular weight fractions can be obtained as aresult, a low molecular weight, a middle molecular weight, and a highmolecular weight fraction. A block copolymer can then be synthesized bya ring opening polymerization of D,L-lactide from the terminal hydroxylgroup of the poly(IPAAm-co-DMAAm) of the middle molecular weightfraction. The resulting poly(IPAAm-co-DMAAm)-b-poly(D,L-lactide)copolymer can be purified as described in Kohori, F., et al. (1999).Control of adriamycin cytotoxic activity using thermally responsivepolymeric micelles composed ofpoly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(D,L-lacide).Colloids Surfaces B: Biointerfaces 16: 195-205.

The compounds of the invention can be loaded into the inner cores ofmicelles and the micelles prepared simultaneously by a dialysis method.For example, a chloride salt of an NDGA derivative can be dissolved inN,N-dimethylacetamide (“DMAC”) and added by triethylamine (“TEA”). Thepoly(IPAAm-co-DMAAm)-b-poly(D,L-lactide) block copolymer can bedissolved in DMAC, and distilled water can be added. The solution ofNDGA derivative and the block copolymer solution can be mixed at roomtemperature, followed by dialysis against distilled water using adialysis membrane with 12,000-14,000 molecular weight cut-off(Spectra/Por®2, spectrum Medical Indus., CA. U.S.A.) at 25° C.Poly(IPAAm-co-DMAAm)-b-poly(D,L-lactide) micelles incorporatingderivatives can be purified by filtration with a 20 nm pore sizedmicrofiltration membrane (ANODISC™, Whatman International), as describedin Kohori, F., et al. (1999).

Preparation of Multivesicular Liposomes Containing NDGA Compounds

Multivesicular liposomes (“MVL”) can be produced by any methodconventional in the art, such as, for example, the double emulsificationprocess as described in Mantriprgada, S. (2002) A lipid based depot(DepoFoam® technology) for sustained release drug delivery. Prog LipidRes. 41: 392-406.

Formulation for Oral Delivery

The compounds of Scheme 1 are water-soluble, hydrophilic compounds, andcan be readily formulated for oral delivery. Compounds of Scheme 2 arehydrophobic and can be formulated in suitable lipophilic solvents knownin the art. The compounds can be formulated with a pharmaceuticallyacceptable carrier or excipient and delivery of such as oralformulations, such as in the form of an aqueous liquid solution of thecompound, or the compounds can be lyophilized and delivered as a powder,or made into a tablet, or the compounds can be encapsulated. Tablets canbe enteric coated tablets, with formulations for sustained release,either slow release or rapid release formulations. Various othercarriers, excipients and adjuvants could be used as are well known tothose skilled in pharmaceutical preparations. The amount of thecompounds to be included in the oral formulations can be adjusteddepending on the desired dose to be administered to a subject. Such anadjustment is within the skill of persons conventional in the art.

Intra-Arterial Administration

The present invention includes formulation of the compounds forintra-arterial administration as is conventional in the art, asdescribed in, for example, Doolittle, N. D. et al. (2000) Cancer 88(3):637-647 and Cloughesy, T. F. et al. (1997) J. Neurononcol. 35: 121-131,with or without accompanying blood brain barrier disruption (“BBBD”),and with or without occlusion, such as in hepatic arterychemoemobolization, as described in Drougas, J. G. et al. (1998) Hepaticartery chemoembolization for management of patients with advancedmetastatic carcinoid tumors. Am. J. Surg. 175: 408-412 and Desai, D. C.et al. (2001) Serum pancreastatin levels predict response to hepaticartery chemoembolization and somatostatin analogue therapy in metastaticneuroendocrine tumors. Reg Peptides 96: 113-117. Briefly, wherecompounds of the invention are administered intra-arterially withocclusion, primary arteries leading to the target site are catheterizedand the compounds are administered through a catheter. Embolization ofthe arteries, in order to retain the compounds at the target site for alonger period, is performed using polyvinyl alcohol particles alone orin combination with coils. Intra-arterial delivery of the compounds islimited to water soluble compositions, and accordingly, the compoundsdisclosed herein will be advantageous for such delivery. The drugs oragents herein can be dissolved in saline prior to intra-arterialinjection and such injection may be preceded by heparin treatment andsedation. For safest treatment of brain tumor, preferably,intra-arterial administration is conducted before tumor burden becomesexcessive.

Osmotic disruption of the blood brain barrier (“BBB”) as conventional inthe art may accompany intra-arterial delivery of the agents herein asdescribed in, for example, Doolittle, N. D. et al. (2000); Sato, S. etal., Acta Neurochir (Wien) 140: 1135-1141; disc 1141-1132 (1998); andBhattacharjee, A. K. et al. Brain Res Protocol 8: 126-131 (2001). Such aprocedure can be used to increase the transfer of drugs into the centralnervous system (“CNS”) preferably just prior to intra-arterial delivery.For such disruption, a catheter is placed into an artery, usually thesuperficial temporal artery, leading to the brain and the BBB isdisrupted with a solution of mannitol. This invasive procedure istypically performed while the patient is under general anesthesia. Suchtreatment may require prior hydration and administration ofanticonvulsants and/or atropine.

Formulation of NDGA Compounds for Intranasal Delivery

The present invention includes formulations of the compounds of theinvention, for intranasal delivery and intranasal delivery. Intranasaldelivery may advantageously build up a higher concentration of theactive agents in the brain than can be achieved by intravenousadministration. Also, this mode of delivery avoids the problem of firstpass metabolism in the liver and gut of the subject receiving the drug.

The amount of the active agent that can be absorbed partly depends onthe solubility of the drug in the mucus, a composition that consists ofabout 95% water solution of serum proteins, glycoproteins, lipids andelectrolytes. Generally, as lipophilicity of the active agents hereinincreases, the drug concentration in the CSF also increases. See, forexample, Minn, A. et al. (2002). Drug transport into the mammalianbrain: the nasal pathway and its specific metabolic barrier. J. DrugTarget, 10: 285-296.

The compounds can be dissolved in a pharmaceutically acceptable carriersuch as saline, phosphate buffer, or phosphate buffered saline. In oneembodiment, a 0.05 M phosphate buffer at pH 7.4 can be used as thecarrier, as described in, for example, Kao, H. D., et al. (2000).Enhancement of the Systemic and CNS Specific Delivery of L-Dopa by theNasal Administration of Its Water Soluble Prodrugs, Pharmaceut. Res.,17(8): 978-984.

Intranasal delivery of the present compounds may be optimized byadjusting the position of the subject when administering the agents. Forexample, the head of the patient may be variously positionedupright-90°, supine-90°, supine-45°, or supine-70°, to obtain maximaleffect.

The carrier included in the present compositions may be any materialthat is pharmaceutically acceptable and compatible with the activeagents of the composition. Where the carrier is a liquid, it can behypotonic or isotonic with nasal fluids and within the pH of about 4.5to about 7.5. Where the carrier is in powdered form it is also within anacceptable pH range.

The carrier composition for intranasal delivery may optionally containlipophilic substances that may enhance absorption of the active agentsacross the nasal membrane and into the brain via the olfactory neuralpathway. Examples of such lipophilic substances include, but are notlimited to, gangliosides and phosphatidylserine. One or severallipophilic adjuvants may be included in the composition, such as, in theform of micelles.

The pharmaceutical composition of active agents for intranasal deliveryto a subject for treatment of tumor and other proliferative diseases,disorders, or conditions herein can be formulated in the mannerconventional in the art as described in, for example, U.S. Pat. No.6,180,603. For example, the composition herein can be formulated as apowder, granules, solution, aerosol, drops, nanoparticles, or liposomes.In addition to the active agents, the composition may containappropriate adjuvants, buffers, preservatives, salts. Solutions such asnose drops may contain anti-oxidants, buffers, and the like.

Delivery by Implantation

The disclosed compounds and compositions, may be delivered to a subjectfor treatment by surgical implantation into a tumor site, with orwithout surgical excision of the tumor, such as by implantation of abiodegradable polymer containing the compounds. In one embodiment, thismethod of treatment can be performed, for example, after surgicalresection, such as in the treatment and resection of brain tumor, asdescribed in, Fleming, A. B. and Saltzman, W. M., Pharmacokinetics ofthe Carmustine Implant, Clin. Pharmacokinet, 41: 403-419 (2002). Thismethod of delivery is applicable to not only brain tumors but to othertumors as well. This treatment may be combined with other conventionaltherapy besides or in addition to surgery, such as radiotherapy,chemotherapy or immunotherapy.

Thus, the biodegradable polymer herein can be any polymer or copolymerthat would dissolve in the interstitial fluid, without any toxicity oradverse effect on host tissues. Preferably, the polymer or monomers fromwhich the polymer is synthesized is approved by the Food and DrugAdministration for administration into humans. A copolymer havingmonomers of different dissolution properties is preferred so as tocontrol the dynamics of degradation, such as increasing the proportionof one monomer over the other to control rate of dissolution.

In one embodiment, the polymer is a copolymer of1,3-bis-(p-carboxyphenoxy)propane and sebacic acid [p(CPP:SA)], asdescribed in Fleming A. B. and Saltzman, W. M., Pharmacokinetics of theCarmustine Implant, Clin. Pharmacokinet, 41: 403-419 (2002); and Brem,H., and Gabikian, P. (2001). Biodegradable polymer implants to treatbrain tumors. J. Control. Rel. 74: 63-67. In another embodiment, thepolymer is a copolymer of polyethylene glycol (“PEG”) and sebacic acid,as described in Fu, J. et al. (2002). New Polymeric Carriers forControlled Drug Delivery Following Inhalation or Injection.Biomaterials, 23: 4425-4433.

Polymer delivery systems are also applicable to delivery of compoundsdisclosed herein. The compounds are combined with the biodegradablepolymers and surgically implanted at the tumor site. Some polymercompositions are also usable for intravenous or inhalation therapyherein.

Delivery Through Inhalation

The compounds disclosed herein may be delivered systemically and/orlocally by administration to the lungs through inhalation. Inhalationdelivery of drugs has been well accepted as a method of achieving highdrug concentration in the pulmonary tissues without triggeringsubstantial systemic toxicity, as well as a method of accomplishingsystemic circulation of the drug. The techniques for producing suchformulations are conventional in the art.

For pulmonary delivery via inhalation, the compounds herein may beformulated into dry powders, aqueous solutions, liposomes,nanoparticles, or polymers and administered, for example, as aerosols.Hydrophilic formulations may also be taken up through the alveolarsurfaces and into the bloodstream for systemic applications.

In one embodiment, the polymers containing the disclosed compounds aremade and used as described in Fu, J. et al. (2002). For example, thepolymers herein can be polymers of sebacic acid and polyethylene glycol(“PEG”), or can be polylactic-co-glycolic) acid (“PLGA”), or polymers ofpolyethyleneimine (“PEI”) and poly-L-lysine (“PLL”).

In another embodiment, the compounds for inhalation delivery may bedissolved in saline or ethanol before nebulization and administered, asdescribed in Choi, W. S. et al. (2001). Inhalation delivery of proteinsfrom ethanol suspensions. Proc. Natl. Acad. Sci. USA, 98(20):11103-11107.

In a further embodiment, the compounds herein are also effective whendelivered as a dry powder, prepared in the manner conventional in theart, as described in, for example, Patton, J. S. et al., InhaledInsulin, Adv. Drug Deliv. Rev., 35: 235-247 (1999).

The present invention includes delivery of the compounds with the aid ofmicroprocessors embedded into drug delivery devices, such as, forexample, SmartMist™ and AERx™, as described in, for example, Gonda, I.,et al. (1998). Inhalation delivery systems with compliance and diseasemanagement capabilities. J. Control. Rel. 53: 269-274.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

The present invention will now be described in greater detail withreference to the following specific, non-limiting examples.

EXAMPLES Example 1meso-2,3-Dimethyl-1,4-bis(3,4-[2-(pyrrolidino)ethoxyphenyl])butaneTetrakishydrochloride Salt (5a)

The Standard Procedure (above) was carried out using NDGA (1, 1.43 g,4.73 mmol, 1.0 equiv), potassium carbonate (6.53 g, 47.3 mmol, 10.0equiv), acetone (150 mL) and 1-(2-chloroethyl)pyrrolidine hydrochloride(4.03 g, 23.7 mmol, 5.0 equiv) to give pure 5a (2.45 g, 3.54 mmol) asyellow gum in 75% yield: ¹H NMR (CDCl₃, 400 MHz) δ 0.77 (d, J=6.4 Hz,6H, 2×CH₃), 1.68 (m, 2H, 2×CH), 1.74-1.77 (m, 16H, 8× pyrrolidine CH₂),2.20 (dd, J=13.6, 9.6 Hz, 2H, 2×ArCH), 2.61-2.66 (m, 16H, 8× pyrrolidineNCH₂), 2.72-2.75 (m, 2H, 2×ArCH), 2.88 (t, J=12.2 Hz, 8H, 4×CH₂N), 4.08(t, J=5.6 Hz, 8H, 4×CH₂O), 6.61-6.63 (m, 4H, 4×ArH), 6.76 (d, J=8.4 Hz,2H, 2×ArH); ¹³C NMR (CDCl₃, 100 MHz) δ 16.01, 23.42, 38.83, 39.39,53.36, 54.63, 54.92, 68.16, 68.27, 113.86, 114.99, 121.45, 134.98,146.78, 148.49; IR KBr 3429 (s), 2959 (m), 2709 (m), 1635 (w), 1515 (m),1457 (m), 1263 (s), 1229 (m), 1141 (s), 1018 (m) cm⁻¹; MS (FAB) m/e(relative intensity) 691 (M+, 41), 594 (7), 592 (6), 523 (3), 496 (3),399 (12), 220 (4), 98 (100), 84 (81), 56 (8); HRMS (FAB) calcd forC₄₂H₆₆N₄O₄ 690.5084, found 691.5088.

Example 2meso-2,3-Dimethyl-1,4-bis(3,4-[2-(piperidino)ethoxyphenyl])butaneTetrakishydrochloride Salt (5b)

In order to make this compound, the non-salt base compound of thecompound referred to herein as P₄N, the Standard Procedure (above) wascarried out using NDGA (1, 1.11 g, 3.67 mmol, 1.0 equiv), potassiumcarbonate (5.07 g, 36.72 mmol, 10.0 equiv), acetone (150 mL) and1-(2-chloroethyl)piperidine monohydrochloride (3.38 g, 18.35 mmol, 5.0equiv) to give pure 5b (1.93 g, 2.46 mmol) as white solids in 67% yield:¹H NMR (CDCl₃, 400 MHz) δ 0.78 (d, J=6.4 Hz, 6H, 2×CH₃), 1.38-1.45 (m,8H, 4× piperidine CH₂), 1.42-2.58 (m, 16H, 8× piperidine CH₂), 1.55-1.68(m, 2H, 2×CH), 2.36-2.51 (m, 16H, 8× piperidine CH₂N), 2.64 (dd, J=13.2,1.2 Hz, 2 H, 2×ArCH), 2.71 (t, J=6.0 Hz, 8H, 4×CH₂N), 4.03 (t, J=2.4 Hz,8H, 4×CH₂O), 6.75 (d, J=7.9 Hz, 2H, 2×ArH), 6.63 (s, 2H, 2×ArH), 6.75(dd, J=7.9, 1.6 Hz, 2H, 2×ArH); ¹³C NMR (CDCl₃, 100 MHz) δ 15.95, 24.00,25.82, 38.82, 39.21, 54.81, 54.87, 57.81, 67.03, 67.18, 113.80, 114.90,121.28, 134.78, 146.69, 148.41; IR KBr 3429 (s), 2951 (m), 2687 (m),1633 (w), 1515 (m), 1455 (m), 1262 (s), 1140 (s), 1017 (m) cm⁻¹; MS(FAB) m/e (relative intensity) 747 (M+, 63), 636 (8), 634 (4), 524 (3),413 (5), 374 (4), 346 (3), 241 (6), 128 (5), 112 (100), 98 (63), 84(15), 56 (7); HRMS (FAB) calcd for C₄₆H₇₄N₄O₄ 746.5710, found 747.5713.

Unless otherwise indicated herein, “P₄N” refers to the HCl salt of thiscompound.

Example 3meso-2,3-Dimethyl-1,4-bis(3,4-[2-(morpholino)ethoxyphenyl])butaneTetrakishydrochloride Salt (5c)

The Standard Procedure (above) was carried out using NDGA (1, 1.19 g,3.94 mmol, 1.0 equiv), potassium carbonate (5.43 g, 39.36 mmol, 10.0equiv), acetone (150 mL), and 4-(2-chloroethyl)morpholine hydrochloride(3.67 g, 19.7 mmol, 5.0 equiv) to give pure 5c (2.46 g, 3.26 mmol) aswhite solids in 83% yield: ¹H NMR (CDCl₃, 400 MHz) δ 0.72 (d, J=6.4 Hz,6H, 2×CH₃), 1.62-1.68 (m, 2H, 2×CH), 2.16 (dd, J=13.4, 9.2 Hz, 2H,2×ArCH), 2.72 (dd, J=13.2, 4.6 Hz, 2H, 2×ArCH), 2.41-2.59 (m, 16H, 8×morpholine CH₂), 2.70 (t, J=12.2 Hz, 8H, 4×CH₂N), 3.46-3.72 (m, 16H, 8×morpholine CH₂), 4.01 (t, J=5.6 Hz, 8H, 4×CH₂O), 6.57-6.58 (m, 4H,4×ArH), 6.72 (d, J=8.4 Hz, 2H, 2×ArH); ¹³C NMR (CDCl₃, 100 MHz) δ 15.88,38.51, 39.03, 53.21, 53.79, 57.41, 59.82, 66.46, 66.56, 66.69, 113.65,114.70, 121.36, 134.84, 146.41, 148.14; IR KBr 3419 (s), 2956 (m), 2606(m), 1635 (w), 1515 (m), 1456 (m), 1264 (s), 1139 (s), 1104 (m), 1041(m), 917 (w) cm⁻¹; MS (FAB) m/e (relative intensity) 755 (M+, 3), 263(2), 236 (3), 149 (8), 114 (100), 100 (67), 56 (18), 42 (10); HRMS (FAB)calcd for C₄₂H₆₆N₄O₈ 754.4881, found 755.4885.

Example 4meso-2,3-Dimethyl-1,4-bis(3,4-[3-(morpholino)propoxyphenyl])butaneTetrakishydrochloride Salt (5d)

The Standard Procedure (above) was carried out using NDGA (1, 1.69 g,5.59 mmol, 1.0 equiv), potassium carbonate (4.63 g, 33.5 mmol, 6.0equiv), acetone (50 mL), and 4-(3-chloropropyl)morpholine hydrochloride(4.55 g, 27.9 mmol, 5.0 equiv) to give pure 5d (3.70 g, 3.88 mmol) aswhite solids in 69% yield: ¹H NMR (D₂O, 400 MHz) δ 0.80 (d, J=6.4 Hz,6H, 2×CH₃), 1.73-1.77 (m, 2H, 2×CH), 2.24-2.38 (m, 8H, 4×CH₂), 2.33 (dd,J=13.6, 9.6 Hz, 2H, 2×ArCH), 2.74 (dd, J=13.6, 5.2 Hz, 2H, 2×ArCH), 3.19(t, J=12.4 Hz, 8H, 4×CH₂N), 3.35-4.12 (m, 32H, 16× morpholine CH₂), 4.18(t, J=5.2 Hz, 8H, 4×CH₂O), 6.84 (dd, J=8.0, 1.6 Hz, 2H, 2×ArH), 6.92 (d,J=1.6 Hz, 2H, 2×ArH), 7.02 (d, J=8.0 Hz, 2H, 2×ArH); ¹³C NMR (CDCl₃, 100MHz) 816.63, 24.26, 39.02, 39.87, 52.73, 55.52, 64.73, 66.93, 67.26,115.26, 116.11, 123.33, 137.43, 146.41, 148.19; IR (KBr) 3441 (s), 2934(s), 2870 (m), 2607 (m), 2474 (m), 1642 (w), 1510 (m), 1425 (m), 1220(s), 1159 (m), 1107 (s), 985 (m), 896 (w) cm⁻¹; MS (FAB) m/e (relativeintensity) 811 (M+, 40), 684 (10), 557 (3), 406 (100), 343 (8), 271(39), 203 (3), 128 (8); HRMS (FAB) calcd for C₄₆H₇₄N₄O₄, 810.5506, found810.5491.

Example 5 meso-2,3-Dimethyl-1,4-bis[3,4-(phenylpiperidine-1-carboxylate)]butane (6a)

To a solution of NDGA (1, 453 mg, 1.49 mmol, 1.0 equiv) in pyridine (20mL) stirred at 0° C. was added 1-piperidinecarbonyl chloride (1.32 g,8.94 mmol, 6.0 equiv). After the reaction mixture was stirred at roomtemperature for 24 h, it was quenched with water and the remainingpyridine was removed under reduced pressure. The resultant was extractedwith CH₂Cl₂ (3×50 mL) and the combined organic layers were washed withsaturated brine, dried over CaCl_(2(s)), filtered, and concentratedunder reduced pressure. The residue was purified by use of columnchromatography (50% EtOAc in hexane as eluant) to give 6a (797 mg, 1.07mmol) in 72% yield as white solids with purity >99.5% as checked by GC:mp (recrystallized from EtOAc) 171.5-172.4° C.; TLC R_(f) 0.51 (50%EtOAc in hexane as eluant); ¹H NMR (CDCl₃, 400 MHz) δ 0.81 (d, J=6.4 Hz,6H, 2×CH₃), 1.58-1.68 (m, 24H, 12×CH₂), 1.72-1.78 (m, 2H, 2×CH), 2.27(dd, J=13.2, 10.2 Hz, 2H, 2×ArCH), 2.76 (dd, J=13.6, 3.6 Hz, 2H,2×ArCH), 3.49-3.56 (m, 16H, 8×CH₂N), 6.95 (dd, J=8.2, 2.0 Hz, 2H,2×ArH), 7.02 (s, 2H, 2×ArH), 7.08 (d, J=8.2 Hz, 2H, 2×ArH); ¹³C NMR(CDCl₃, 100 MHz) δ 16.00, 24.14, 24.31, 25.90, 39.03, 39.73, 45.12,45.46, 122.94, 123.86, 126.32, 139.71, 141.15, 142.86, 152.93, 153.02;IR (KBr) 3425 (m), 2933 (s), 2858 (m), 1729 (s), 1509 (m), 1418 (s),1376 (w), 1351 (w), 1251 (s), 1235 (s), 1197 (s), 1139(s), 1118 (s),1048 (w), 1023 (m), 894 (w), 852 (w), 794 (w), 749 (w) cm⁻¹; MS (EI) m/e(relative intensity) 747 (M+, 5), 702 (2), 674(1), 635 (6), 575(17), 505(18), 373 (5), 112 (100), 84 (3), 69 (20); HRMS (EI) calcd forC₄₂H₅₈N₄O₈, 746.4255, found 746.4253.

Example 6 meso-2,3-Dimethyl-1,4-bis[3,4-(methyl phenyl carbonate)]butane(6b)

To a solution of NDGA (1, 1.07 g, 3.53 mmol, 1.0 equiv) in pyridine (50mL) at 0° C. was added methyl chloroformate (3.33 g, 35.27 mmol, 10.0equiv). After the reaction mixture was stirred at room temperature for24 h, it was quenched with water and pyridine was removed under reducedpressure. The resultant was extracted with CH₂Cl₂ (3×50 mL) and thecombined organic layers were washed with saturated brine, dried overCaCl_(2(s)), filtered, and concentrated under reduced pressure. Theresidue was purified by column chromatography on silica gel (50% EtOAcin hexane as eluant) to give 6b (1.57 g, 2.93 mmol) in 83% yield aswhite solids with purity >99.5% as checked by GC: mp (recrystallizedfrom EtOAc) 101.6-102.4° C.; TLC R_(f) 0.63 (50% EtOAc in hexane aseluant); ¹H NMR (CDCl₃, 400 MHz) δ 0.83 (d, J=6.4 Hz, 6H, 2×CH₃),1.76-1.78 (m, 2H, 2×CH), 2.32 (dd, J=13.2, 9.2 Hz, 2H, 2×ArCH), 2.76(dd, J=13.2, 4.6 Hz, 2H, 2×ArCH), 3.90 (s, 6H, 2×CH₃O), 3.91 (s, 6H,2×CH₃O), 6.99-7.03 (m, 4H, 4×ArH), 7.16 (d, J=8.4 Hz, 2H, 2×ArH); ¹³CNMR (CDCl₃, 100 MHz) δ 16.31, 39.03, 39.52, 55.91, 122.84, 123.63,127.53, 140.55, 141.09, 142.23, 153.67; IR (KBr) 2971 (m), 2915 (w),1772 (s), 1508 (m), 1446 (m), 1379 (w), 1197 (s), 1131 (m), 1048 (m),931 (m), 824 (w), 810 (w), 783(w) cm⁻¹; MS (EI) m/e (relative intensity)534 (M+, 13), 490 (9), 459 (5), 400 (4), 195 (100), 151 (70), 137 (19),105 (13), 91 (7), 77 (9); HRMS (EI) calcd for C₂₆H₃₀O₁₂, 534.1737, found534.1739.

Example 7 meso-2,3-Dimethyl-1,4-bis(benzo[d][1,3]-dioxol-2-one)butane(7)

To a solution of NDGA (1, 1.26 g, 4.15 mmol, 1.0 equiv) in THF (50 mL)at room temperature was added 1,1′-carbonyldiimidazole (2.69 g, 16.61mmol, 4.0 equiv). After the reaction mixture was stirred at roomtemperature for 24 h, it was quenched with water and THF was removedunder reduced pressure. The resultant was extracted with CH₂Cl₂ (3×50mL) and the combined organic layers were washed with saturated brine,dried over CaCl_(2(s)), filtered, and concentrated under reducedpressure. The residue was purified by column chromatography on silicagel (20% EtOAc in hexane as eluant) to give 7 (1.11 g, 3.13 mmol) in 74%yield as white solids with purity >99.5% as checked by GC: mp(recrystallized from EtOAc) 172.6-173.4° C.; TLC R_(f) 0.59 (20% EtOAcin hexane as eluant); ¹H NMR (CDCl₃, 400 MHz) δ 0.83 (d, J=6.8 Hz, 6H,2×CH₃), 1.72-1.78 (m, 2H, 2×CH), 2.37 (dd, J=13.2, 9.6 Hz, 2H, 2×ArCH),2.76 (dd, J=13.6, 4.4 Hz, 2H, 2×ArCH), 6.97 (dd, J=8.2, 1.2 Hz, 2H,2×ArH), 7.02 (s, 2H, 2×ArH), 7.13 (d, J=8.2 Hz, 2H, 2×ArH); ¹³C NMR(CDCl₃, 100 MHz) δ 16.18, 39.64, 39.93, 110.17, 110.90, 125.34, 139.25,141.68, 143.60, 151.59; IR (KBr) 2991 (s), 1821 (s), 1638 (w), 1488 (m),1438 (m), 1384 (m), 1347 (m), 1322 (w), 1239 (s), 1152 (m), 1044 (m),965 (s), 931 (m), 882 (w), 832 (w), 786 (w), 757(m), 711(w) cm⁻¹; MS(EI) m/e (relative intensity) 354 (M+, 24), 298 (1), 205 (36), 189 (6),163 (24), 149 (100), 105 (37), 91 (8), 77 (32), 65 (5), 51 (9); HRMScalcd for C₂₀H₁₈O₆, 354.1103, found 354.1108.

Example 8 The Secreted Alkaline Phosphatase (SeAP) Assay

The screening of the new NDGA derivatives for inhibition of Tattransactivation was achieved with SeAP assay originally described byBerger et al.^(12,13,19) The COS cells were maintained in Iscove'sModified Dulbecco's Medium supplemented with 10% (v/v) fetal serum, 100unit/mL penicillin, 0.25 μg/mL Fungizone, and 100 μg/mL streptomycin.The medium used for dilution of compounds and maintenance of culturesduring the assay was the same as described above. Cultures weremaintained in disposable tissue culture labware at 37° C. in ahumidified atmosphere of CO₂ (5.0%) in air. Compounds were dissolved asstock solutions in water or dimethyl sulfoxide (DMSO) at theconcentration of 10 mM. The stock solutions were diluted with H₂O (5a-d)or DMSO (6a,b and 7) and dilute by medium to the desired concentrationby a 30-s vortex just before addition into the cell culture. Triplicatecell samples were seeded at density of ˜1.5×10⁴ cell per well in Libro24-well flat-bottom culture dishes (17-mm diameter), which wereincubated for 24 h until they reached 50% confluence. The cells werecotransfected by the calcium phosphate procedure with DNA from plasmidspBC12/CMV/t2 (coding for Tat function, 0.20 μg/well) and pBC12/HIV/SeAP(0.40 μg/well). Cells and DNAs were kept in contact for 6.0 h; then themedium was aspirated and replaced by 500 μL of medium containing thetest compound. The compound-treated cells were then incubated foradditional 48 h. At the end of incubation, an aliquot of cell culturemedium was removed and SeAP activities were analyzed by spectrametricmethod.²⁰ The sample of culture medium were heated at 65° C. for 5.0 minto inactivate endogenous phosphatases selectively (SeAP is thermalstable). The buffer solution of 2×SeAP (100 μL, 1.0 M diethanolamine, pH9.8; 0.50 mM MgCl₂; 10 mM L-homoarginine) was added to the culturemedium sample (100 μL) in a 96-well flat-bottom culture dish (Corning).Then, the substrate solution (20 mL, 120 mM, p-nitrophenyl phosphatedissolved in 1×SeAP buffer) was dispensed into each well containing thereaction mixture. The absorbance at wavelength 405 nm specific for thehydrolysis product was read at 2.5 min intervals at 37° C. over thecourse of 60 min on an EL340i microplate reader (Bio-tek Instruments)with 5 s automatic shaking before each reading. The percent inhibitionof SeAP expression was calculated at 60 min.^(12,13) This assay was usedin Example 11, below.

Example 9 Cytotoxicity Assay

The cytotoxicity of P₄N against Vero cells and human cancer cells HEP3B,HT29 and MCF7 was analyzed using an MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]-basedassay (Sigma). The absorbance of OD₅₇₀ was a measurement of formazoncrystals solubilized in DMSO from metabolically active cells. Procedureswere discussed in Huang R C C, et al. in Antiviral Res 58, 57-64 2003.¹⁰

Example 10 Interaction of P₄N with Double Stranded Deoxy OligonucleotideContaining Sp1 Binding Site

We have shown in crystallographic studies that the presence of the drugP₄N alters the conformation of dsOLIGO_(sp) by the opening of the majorgroove and exposure of the minor groove, two features of the structureof the DNA that are critical for the binding of Sp1. These structuralchanges in the grooves of the DNA are attributed to the P₄N-inducedchanges in the global conformation of the DNA (FIG. 2).²¹ The opening orwidening of the groove alters the conformation of the dsOLIGO_(sp),thereby inhibiting or preventing the binding of Sp1. Accordingly, P₄Nand the other compounds of the invention should be useful for treatingdiseases and disorders wherein Sp1 binding involved in the diseaseprocess (e.g. in viral replication or tumor growth).

The titration of the Sp1 cognate binding site with P₄N was furtherstudied by 1D NMR (FIG. 3). The drug binds to the DNA in the micro-molarrange. Changes in the chemical shifts and the line-widths of both theDNA and the drug indicate structural perturbations of the DNA upontitration with P₄N. The 2D ¹H-¹³C HSQC data (FIG. 4) are consistent withP₄N adopting two different conformations and/or exhibiting two differentmodes of binding. This is reflected in the split of the well resolvedresonances of the carbon-nitrogen ring CH₂ protons (which lie in an areaof the spectrum that is free of DNA peaks). As shown on both FIG. 3 andFIG. 4 the largest perturbations of the P₄N chemical shifts induced uponbinding are those of the CH₃ protons, aromatic protons and thecarbon-nitrogen ring CH₂ protons. The NMR results are consistent (or inagreement) with the results from the UV-melts which unequivocallydemonstrate that P₄N binds to its Sp1 cognate binding site with theformation of at least two distinct complexes (FIG. 5). Binding of Sp1 todsOLIGO_(sp) and binding of P₄N to dsOLIGO_(sp) are both reversible asshown in a competitive band-shift analysis (FIG. 6).

We have also performed graphic molecular modeling of P₄N withdsOLIGO_(sp) using the programs “Builder and Biopolymer for Constructionof Structures”. The energies for conformations were minimized by the useof the program “Discover with the Consistent Valence Forcefield (CVFF).Compound 5b, P₄N binds the major groove of the Sp1 cognate binding sitewith a ΔE of −4506.77 KCal/Mole. P₄N on the other hand, was found to beexpelled from the minor groove of the dsOLIGO_(sp) because of stericcongestion (FIG. 7).

Example 11 Inhibition of HIV Tat-Regulated Transactivation by NDGADerivatives

Two plasmid constructs¹⁹ were used to test the effect of synthesizedNDGA derivatives on Tat-regulated HIV transactivation as describedpreviously.^(12,13,19) The plasmid constructs included a cytomegalovirus(CMV) promoter driven tat gene and SP1 regulated HIV LTR promoter drivenreporter gene (i.e., SeAP). A standard SeAP assay in the absence of drugwas initially run to find out efficiency of transfection. Results, shownin FIG. 8, indicate a nearly 5 fold increment in Tat-induced SeAPexpression after 60 min in comparison with the control levels of theenzyme activity (i.e. without DNA transfection or transfection withHIV/SeAP alone). Both of them were close to the baseline.

We tested the NDGA derivatives 5a-d, 6a,b, and 7 with the SeAP assay, ofwhich the results displayed a dose-dependent inhibitory activity of Tattransactivation (see Table 1). At 80 μM, all of these NDGA derivativesinhibited the Tat-regulated SeAP production to the level >90% andexhibited a greater inhibitory activity than the parent NDGA (1, IC₅₀=20μM) and Mal.4 (3, IC₅₀=25 μM).¹² Except 5a (IC₅₀ 17.2 μM) and 5c (IC₅₀17.3 μM), all other NDGA derivatives of these new series also showedgreater potency than M₄N (2, IC₅₀=11.1 μM).⁹ P₄N showed no effect on thegrowth of the cos cells≦1 μM, yet at this concentration HIVtransactivation was fully inhibited (FIG. 9). In fact, P₄N is thestrongest antiviral and anticancer inhibitor of all of the NDGAderivatives that we have developed to date.

TABLE 1 Inhibition (%) of HIV Tat-regulated Transactivation in COS Cellsby NDGA derivatives. Concentration (μM) ^(a) IC₅₀ ^(b) Compound 0.100.25 0.50 1.0 2.5 5.0 10 20 40 80 100 (μM) 5a 18.1 15.4 27.2 19.6 31.014.1 26.3 59.1 94.8 97.1 100 17.23 5b 8.7 4.3 29.0 56.8 90.6 97.0 99.8100 100 100 97.7 0.88 5c 5.0 16.5 9.9 8.1 4.6 13.3 25.4 58.9 86.0 96.8100 17.34 5d 15.0 15.9 10.6 9.5 52.7 89.5 96.0 99.9 99.8 100 89.5 2.416a 0 3.5 5.8 11.7 46.2 78.6 84.0 86.1 83.8 90.1 89.8 2.79 6b 9.8 12.316.0 13.1 15.1 38,0 51,6 58.4 70.8 93.4 88.9 9.41 7 7.5 18.1 4.2 5.923.7 47.1 51.2 68.8 85.7 96.3 91.6 8.54 ^(a) All data represent theaverage of three experiments. ^(b) Concentrations exhibiting 50%inhibitory (IC₅₀) represented the mean of the triplicate determinationswith standard deviations.

Example 12 P₄N as Anti-HSV Agent in a Mouse Vaginal Model

The efficacies of P₄N against HSV-2 in a mouse vaginal model system havebeen examined. Progestin treated female CF-1 mice (10 per group) wereinfected with HSV-2 in the absence or presence of P₄N (in PBS). At theend of three days, the vaginal lavages were collected from the washingsof the vaginal regions. The status of the vaginal HSV-2 infection andreplication were examined by their cytopathic effects on cultured humanforeskin fibroblast cells. Monitoring and scoring were carried out bythree persons independently. P₄N was found to be quite effective. At 10mM concentration only 2 of 10 mice were weakly infected. An averagescale of 0.19 was obtained with little or no toxicity in animals ascompared to an average 2.43 scale when viral infection was proceeded inthe absence of P₄N (FIG. 10).

Example 13 P₄N Inhibits the Growth of Human Tumor Cells in Culture

We have previously shown that M₄N arrests tumor cells growth at G₂-Mphase of the cell cycle by reducing the level of Cdc₂ protein and kinaseactivity,^(17,18) and induces apoptosis by suppressing survivin geneexpression and protein stability and activating the mitochondrialapoptosis pathway.¹⁷ M₄N is able to reverse the multidrug resistance byinhibiting the Sp1-regulated MDR₁ gene expression and preventing thesynthesis of p-glycoprotein (Pgp).²⁴ To investigate the effect of watersoluble NDGA derivative P₄N on growth of the transformed cells, fourcell lines, Vero cell (African green monkey kidney cells), human livercancer line Hep3B, colon cancer cell line HT29, and human breast cancerline MCF-7 were selected for testing. The Vero cell line is aneuploid.Although these cells can grow indefinitely in culture, they generally donot form tumors in immunosuppressed mice, while the three selected humancancer cell lines readily do. P₄N (48 hrs, 37° C.) treatment was foundto be extremely effective in inhibition of all four rapidly dividingtransform cells (FIG. 11) while much less so toward stationary phasevero cells (FIG. 11A).

The presence of P₄N altered the conformation of double strandeddeoxyoligonucleotide containing Sp1 binding site ‘5GGGCGGG3’(dsOLIGO_(sp)) by widening its major groove with an increased opening of2.3 Å (Ref. 25, JMB 349, 731-744 2005). Such P₄N/dsOLIGO_(sp)interactions were further supported by ID and 2D NMR spectroscopy and byuv-melts from the current study. By inducing structural distortion ofSp1 regulated promoters, P₄N makes the cognate site unrecognizable bythe Sp1 protein, a key transcription factor for gene expressions inrapidly dividing systems. In addition to inhibiting HIV-1transactivation, P₄N suppresses HSV-2 replication in a mouse vaginalmodel when treated locally. Proliferations of three human cancer celllines, Hep3B, HT-29 and MCF-7 were effectively stopped by P4N with IC50of 2 μM, 0.8 μM and 1.4 μM respectively.

In comparison with compounds 6a,b and 7, the ether linkage in compounds5a-d resulted in much greater stability in aqueous medium. The resultsof the HIV Tat-regulated transactivation experiments indicated that thepiperidine derivative 5b possessed much greater activity than thepyrrolidine derivative 5a. The angle strain in the five-membered ringsis greater than that in six-membered rings, which may limit thebioactivity. On the other hand, placement of an oxygen atom at the4-position in the six-membered ring decreased the anti-HIVtransactivation activity; the morpholine derivative 5c (IC₅₀=17.34 μM)was less potent than piperidine derivative 5b (IC₅₀=0.88 μM). In themorpholine series (cf. 5c and 5d), we found that activity was increasedby elongating the spacer from two methylene units to three units (Scheme1 and Table 1). Among these NDGA derivatives, compound 5b (P₄N) showedmost potent activity in suppressing HIV Tat-regulated transactivation.It was able to inhibit HSV-2 infection with no apparent sign of toxicityin female mice treated locally with 10 mM (FIG. 10). In addition, P₄Nwas found to effectively eliminate the growth of three human tumor cellsat concentrations <5 μM (FIG. 11).

All publications cited herein are hereby incorporated by reference.

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We claim:
 1. A method of treatment of a disease or disorder, wherein thetreatment does not include prevention, and wherein the disease ordisorder is selected from the group consisting of liver tumor, breasttumor, colon tumor, HIV infection and HSV infection, in a subject inneed thereof, comprising the steps of: (a) providing a compositioncomprising a pharmaceutically acceptable carrier or excipient and atleast one compound of formula

wherein R₁-R₄ are independently selected from the group consisting of:(i) a straight chain or branched lower alkyl group substituted with aheterologous nitrogen-containing ring group of five to seven members;(ii) in combination with the O attached to the phenyl ring, a carbamategroup, other than a methyl carbamate group, optionally containing a fiveor six membered carbon-nitrogen ring; (iii) in combination with the Oattached to the phenyl ring, a C₁-C₆ straight chain or branchedcarbonate group; and (iv) wherein R₁ and R₂; or R₃ and R₄; or R₁ and R₂,and R₃ and R₄; together with the phenyl group to which they areattached, combine to form a cyclic carbonate group; or a salt of thecompound; and (b) administering to the subject an effective amount ofthe composition to treat said disease or disorder.
 2. The method ofclaim 1, wherein the route of administration is selected from the groupconsisting of intranasal administration; oral administration; inhalationadministration; subcutaneous administration; transdermal administration;intradermal administration; intra-arterial administration, with orwithout occlusion; intracranial administration; intraventricularadministration; intravenous administration; buccal administration;intraperitoneal administration; intraocular administration;intramuscular administration; implantation administration; topicaladministration and central venous administration.
 3. The method of claim2, wherein the method comprises administering the composition orally. 4.The method of claim 2, wherein the method comprises administering thecomposition intravenously.
 5. The method of claim 1, wherein thepharmaceutically acceptable carrier or excipient comprises a carrier orexcipient selected from the group consisting of dimethyl sulfoxide(DMSO), phosphate buffered saline, saline, a lipid based formulation, aliposomal formulation, a nanoparticle formulation, a micellarformulation, a water soluble formulation, a biodegradable polymer, anaqueous preparation, a hydrophobic preparation, a lipid based vehicle,and a polymer formulation.
 6. The method of claim 5, wherein thenanoparticle formulation comprises at least one selected from the groupconsisting of poly(DL-lactide-co-glycolide), poly vinyl alcohol,d-α-tocopheryl polyethylene glycol 1000 succinate, andpoly(lactide-co-glycolide)-monomethoxy-poly(polyethylene glycol).
 7. Themethod of claim 5, wherein the liposomal formulation comprises at leastone selected from the group consisting ofphosphatidylcholine/cholesterol/PEG-DPPE,distearoylphosphatidylcholine/cholesterol/PEG-DPPE, and1-2-dioleoyl-sn-glycero-3-phosphocholine/1-2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol)sodium salt/cholesterol/triolein/tricaprylin.
 8. The method of claim 5,wherein the polymer formulation is a biodegradable polymer formulation.9. The method of claim 8, wherein the polymer formulation comprises atleast one ingredient selected from the group consisting of1,3-bis(p-carboxyphenoxy)propane, sebacic acid, poly(ethylene-co-vinylacetate), and poly(lactide-co-glycolide).
 10. The method of claim 1,wherein the pharmaceutically acceptable carrier or excipient allows forhigh local drug concentration and sustained release over a period oftime.
 11. The method of claim 1, wherein the composition is in a formselected from the group consisting of a powder, an aerosol, an aqueousformulation, a liposomal formulation, a nanoparticle formulation, and ahydrophobic formulation.
 12. The method of claim 1, wherein thecomposition is formulated in an orally administrable form selected fromthe group consisting of a tablet, a powder, a gel capsule, a liquid, andan oral rinse.
 13. The method of claim 1, wherein the compound isselected from the group consisting ofmeso-2,3-dimethyl-1,4-bis(3,4-[2-(pyrrolidino)ethoxyphenyl])butane,meso-2,3-dimethyl-1,4-bis(3,4-[2-(piperidino)ethoxyphenyl])butane,meso-2,3-dimethyl-1,4-bis(3,4-[2-(morpholino)ethoxyphenyl])butane,meso-2,3-dimethyl-1,4-bis(3,4-[3-(morpholino)propoxyphenyl])butane,meso-2,3-dimethyl-1,4-bis[3,4-(phenyl piperidine-1-carboxylate)]butane,meso-2,3-dimethyl-1,4-bis[3,4-(methyl phenyl carbonate)]butane, andmeso-2,3-dimethyl-1,4-bis(benzo[d][1,3]dioxol-2-one)butane; and saltsthereof.
 14. The method of claim 1, wherein the method comprisesadministering at least two compounds of formula

of claim 11 in a composition.
 15. The method of claim 14, wherein thetwo compounds are administered substantially contemporaneously.
 16. Themethod of claim 14, wherein the two compounds are administered atdifferent times.
 17. The method of claim 1, wherein the method comprisesadministering the composition more than once.
 18. The method of claim 1,wherein the composition is administered daily for a defined period oftime.
 19. The method of claim 1, wherein the composition is administeredintermittently.
 20. The method of claim 1, wherein the composition isinfused into the subject.
 21. The method of claim 1, wherein thecompound is water soluble.
 22. The method of claim 1, wherein thecomposition is a liquid, an aerosol, an oral rinse, a suspension, atablet, a powder, or a gel capsule.
 23. The method of claim 1, whereinthe compound is administered to a human in an amount selected from thegroup consisting of about 10 mg/kg to about 375 mg/kg per dose; about 10mg/kg to about 250 mg/kg per dose; about 10 mg/kg to about 200 mg/kg perdose; about 10 mg/kg to about 150 mg/kg per dose; about 10 mg/kg toabout 100 mg/kg per dose; about 10 mg/kg to about 75 mg/kg per dose andabout 10 mg/kg to about 50 mg/kg per dose.
 24. The method of claim 1,wherein the composition is administered intravenously.
 25. The method ofclaim 1, wherein the dosage administered results in a concentration in atarget tissue of the subject selected from the group consisting of about0.01 μM to about 100 mM and about 0.01 μM to about 10 mM.
 26. A methodof widening the major groove of a double stranded deoxyoligonucleotidecontaining ‘5GGGCGGG3’ (dsOLIGO_(sp)), thereby making it structurallyunsuitable for Sp1 binding, comprising contacting thedeoxyoligonucleotide with at least one compound of formula

wherein R₁-R₄ are independently selected from the group consisting of:(a) a straight chain or branched lower alkyl group substituted with aheterologous nitrogen-containing ring group of five to seven members;(b) in combination with the O attached to the phenyl ring, a carbamategroup, other than a methyl carbamate group, optionally containing a fiveor six membered carbon-nitrogen ring; (c) in combination with the Oattached to the phenyl ring, a C₁-C₆ straight chain or branchedcarbonate group; and (d) wherein R₁ and R₂; or R₃ and R₄; or R₁ and R₂,and R₃ and R₄; together with the phenyl group to which they areattached, combine to form a cyclic carbonate group; or a salt of thecompound; whereby the major groove is widened.
 27. The method of claim1, wherein the compound inhibits expression of a Sp-1 regulatedeukaryotic gene in a mammalian cell.
 28. The method of claim 1, whereinthe compound inhibits HIV Tat-regulated transactivation in the subject.29. The method of claim 1, wherein the compound widens the major grooveof a double stranded deoxyoligonucleotide containing an Sp1 bindingsite.